Genistein directly blocks glycine receptors of rat neurons freshly isolated from the ventral tegmental area.

The effects of tyrosine kinase inhibitors on the glycine-induced current (I(Gly)) were studied in rat neurons freshly isolated from the ventral tegmental area (VTA). Genistein reversibly and concentration-dependently depressed I(Gly), with an IC(50) of 13 microM. Preincubation with genistein had no effect on I(Gly), indicating that genistein is effective only when glycine is bound to the receptor and channels are most likely open. Genistein depressed maximum I(Gly) without significantly changing the EC(50) for glycine. Genistein-induced inhibition of I(Gly) was sensitive to membrane voltage, being greater at positive membrane potentials. A kinetic analysis indicated that genistein lengthens the time constant of I(Gly) activation, but has no effect on deactivation or desensitization. When genistein was rapidly washed out, a transient rebound current probably reflected a faster dissociation of genistein, with respect to glycine. Results of competition experiments suggest that genistein acts on the same region of the glycine receptor as picrotoxin. Daidzein, an analog of genistein that does not act on protein kinases, also inhibited I(Gly). Co-application of lavendustin A, a specific inhibitor of tyrosine kinase, had no effect on I(Gly). Our results extend to neurons isolated from the VTA, the previous finding that genistein directly inhibits glycine receptors of hypothalamic brain slices.

Decay of ethanol-induced suppression of glycine-activated current of ventral tegmental area neurons.

We demonstrated previously that ethanol depresses glycine-induced currents in 45% of neurons freshly isolated from the ventral tegmental area (VTA) of rats (), and that protein kinase C (PKC) modulates this action of ethanol (). In the present study, we investigated the time course of this effect of ethanol on VTA neurons from young rats. For 70% of the neurons in which ethanol reduced glycine-evoked currents, this depressant effect gradually diminished during continuous superfusion with ethanol. Its action decayed faster when ethanol was applied in several brief pulses than by continuous superfusion. On the other hand, the decay was especially slower when ethanol was applied in pulses at longer intervals or by preincubation. Phorbol ester 12,13-dibutyrate (PDBu, 1 microM), an activator of PKC, also depressed glycine-induced currents. In approximately 40% (6/15) of the neurons, the effect of PDBu diminished with time and was antagonized by the specific PKC inhibitor, chelerythrine (7 microM). Chelerythrine also attenuated the ethanol-induced depression of glycine-induced currents and its time-dependent decay, thus confirming our previous evidence that PKC mediates, at least in part, the decay of the depressant effect of ethanol on glycine-induced currents of VTA neurons.

Moderate hypoglycemia aggravates effects of hypoxia in hippocampal slices from diabetic rats.

We recorded the effects of hypoxia combined with relative hypoglycemia on pre- and post-synaptic potentials in the CA1 area of slices from 4-month-old control and diabetic (streptozotocin-treated) Wistar rats. In experiments on slices kept in 10 or 4 mM glucose (at 33 degrees C), hypoxia was applied until the pre-synaptic afferent volley disappeared--after 12-13 min in most slices, but much earlier (5+/-0.8 min) in diabetic slices kept in 4 mM glucose. When oxygenation was resumed, the afferent volley returned in all slices, for an overall mean recovery of 86.5% (+/-8.8%). Field post-synaptic potentials were fully blocked within 2-3 min of the onset of hypoxia. After the end of hypoxia, they failed to reappear in some slices: overall, their recovery varied between 62 and 68% in control slices, as well as in diabetic slices kept in 10 mM glucose; but recovery was very poor in diabetic slices kept in 4 mM glucose (only 15+/-0.94%). In the latter, hypoxic injury discharges occurred earlier (4.2+/-0.68 min vs. 6.5-8 min for other groups). We conclude that diabetes appears to make hippocampal slices more prone to irreversible loss of synaptic function and early block of axonal conduction when temporary hypoxia is combined with moderate hypoglycemia.

Ethanol inhibition of glycine-activated responses in neurons of ventral tegmental area of neonatal rats.

The brain is particularly sensitive to alcohol during the period of its rapid growth. To better understand the mechanism(s) involved, we studied ethanol effects on glycine-activated responses of ventral tegmental area (VTA) neurons isolated from the newborn rat, using whole cell and gramicidin perforated patch-clamp techniques. Previously we reported that 0.1-40 mM ethanol enhances glycine-induced responses of 35% of VTA neurons. We now direct our attention to the inhibitory effects of ethanol observed in 45% (312 of 694) of neonatal VTA neurons. Under current-clamp conditions, 1 mM ethanol had no effect on the membrane potential of these cells, but it decreased glycine-induced membrane depolarization and the frequency of spontaneous action potentials. Under voltage-clamp conditions, 0.1-10 mM ethanol did not elicit a current but depressed the glycine-induced currents. The ethanol-induced inhibition of glycine current was independent of membrane potential (between -60 and +60 mV). Likewise, ethanol did not alter the reversal potential of the glycine-activated currents. Ethanol-mediated inhibition of glycine current depended on the glycine concentration. While ethanol strongly depressed currents activated by 30 microM glycine, it had no appreciable effect on maximal currents activated by 1 mM glycine. In the presence of ethanol (1 mM), the EC(50) for glycine increased from 32 +/- 5 to 60 +/- 3 microM. Thus ethanol may decrease the agonist affinity of glycine receptors. A kinetic analysis indicated that ethanol shortens the time constant of glycine current deactivation but has no effect on activation. In conclusion, by altering VTA neuronal function, ethanol-induced changes in glycine receptors may contribute to neurobehavioral manifestations of the fetal alcohol syndrome.

Unlike 2-deoxy-D-glucose, 3-O-methyl-D-glucose does not induce long-term potentiation in rat hippocampal slices.

Equimolar replacement of 10 mM glucose by 2-deoxy-D-glucose (2-DG) causes substantial depression followed by a sharp and sustained potentiation of CA1 field EPSPs. In the present experiments, similar applications of 3-O-methyl-D-glucose, which is also taken up by cells but is not phosphorylated, had only a weak blocking action and elicited no potentiation. Possible explanations for the marked effects of 2-DG include a more rapid block of glycolysis and the production of phosphorylated derivatives of 2-DG.

Ethanol potentiation of glycine-induced responses in dissociated neurons of rat ventral tegmental area.

The potentiation of glycine-induced responses by ethanol (EtOH) was studied in neurons freshly dissociated from the ventral tegmental area (VTA) of 5- to 14-day-old postnatal rats using whole-cell and gramicidin-perforated patch-clamp techniques. Under current-clamp conditions, EtOH increased glycine-induced membrane depolarization and action potential firing. Under voltage-clamp conditions, EtOH (0. 1-40 mM) alone did not elicit a current. When coapplied with glycine, EtOH enhanced the glycine-induced current in 35% (180 of 474) of the neurons. The EtOH-induced enhancement of glycine current was independent of membrane potential (between -60 and +60 mV); the reversal potential was not changed. Concentration-response analysis showed that in the presence of EtOH (10 mM), the EC(50) for glycine decreased from 25 +/- 4 to 14 +/- 3 microM; the Hill coefficient increased from 1.5 +/- 0.2 to 1.9 +/- 0.3. Kinetic analysis of glycine currents indicated that EtOH decreased the time constant of activation and increased the time constant of deactivation of glycine-gated chloride channels. EtOH may accelerate glycine association with its receptor at the agonist binding site and increase the apparent agonist affinity. Our observations suggest that, at pharmacologically relevant concentrations, EtOH alters the function of glycine receptors and thus the excitability of neonatal VTA neurons. This action of EtOH may contribute to the neurobehavioral disturbances associated with fetal alcohol syndrome.

2-deoxyglucose induces LTP in layer I of rat somatosensory cortex in vitro.

Temporary replacement of glucose by 2-deoxyglucose (2-DG) induces a long-term potentiation (2-DG-LTP) of excitatory synaptic transmission in hippocampal slices. We therefore examined the effects of 2-DG on monosynaptic field excitatory postsynaptic potentials (fEPSPs) in slices of somatosensory cortex from rats. Monosynaptic fEPSPs were elicited in layer I by stimulating horizontal projections in the same layer. Replacement of glucose (10 mM) in the artificial cerebrospinal fluid with 10 mM 2-DG for 15-17 min produced a minor reduction (by 10-30%), followed by a sustained increase (by approximately 150%) in synaptic responses that could last for over 2 hours. Equimolar replacement of glucose with sucrose did not induce potentiation. The addition of 5 or even 2.5 mM glucose to 10 mM 2-DG largely suppressed the effects of 2-DG; but topically-applied GABA antagonists bicuculline and CGP 35348 did not prevent 2-DG-LTP. Unlike hippocampal 2-DG-LTP, neocortical 2-DG-LTP was: (1) not sensitive to 2-amino-5-phosphonopentanoic acid (AP5); and (2) usually not depotentiated by stimulation at 1 Hz. We conclude that 2-DG produces a robust and sustained increase in synaptic transmission in the neocortex through mechanisms that are independent of NMDA receptor activation.

2-Deoxyglucose-induced long-term potentiation of monosynaptic IPSPs in CA1 hippocampal neurons.

In previous experiments on excitatory synaptic transmission in CA1, temporary (10-20 min) replacement of glucose with 10 mM 2-deoxyglucose (2-DG) consistently caused a marked and very sustained potentiation (2-DG LTP). To find out whether 2-DG has a similar effect on inhibitory synapses, we recorded pharmacologically isolated mononosynaptic inhibitory postsynaptic potentials (IPSPs; under current clamp) and inhibitory postsynaptic currents (IPSCs; under voltage clamp); 2-DG was applied both in the presence and the absence of antagonists of N-methyl-D-aspartate (NMDA). In spite of sharply varied results (some neurons showing large potentiation, lasting for >1 h, and many little or none), overall there was a significant and similar potentiation of IPSP conductance, both for the early (at approximately 30 ms) and later (at approximately 140 ms) components of IPSPs or IPSCs: by 35.1 +/- 10.25% (mean +/- SE; for n = 24, P = 0.0023) and 36.5 +/- 16.3% (for n = 19, P = 0.038), respectively. The similar potentiation of the early and late IPSP points to a presynaptic mechanism of LTP. Overall, the LTP was statistically significant only when 2-DG was applied in the absence of glutamate antagonists. Tetanic stimulations (in presence or absence of glutamate antagonists) only depressed IPSPs (by half). In conclusion, although smaller and more variable, 2-DG-induced LTP of inhibitory synapses appears to be broadly similar to the 2-DG-induced LTP of excitatory postsynaptic potentials previously observed in CA1.

Hypoxia on hippocampal slices from mice deficient in dystrophin (mdx) and isoforms (mdx3cv).

Slices from control C57, mdx, and mdx3cv mice were made hypoxic until both field excitatory postsynaptic potential (fEPSP) and presynaptic afferent volley (AV) disappeared (H1). After reoxygenation and recovery of fEPSP, a second and longer hypoxic test (H2) lasted 3 minutes beyond the time required to block AV. When slices were kept in 10 mmol/L glucose, HI abolished AV 37 and 19% earlier in slices from mdr and mdx3cv mutants than in control slices (where HI = 12 +/- 4.6 minutes, mean +/- SD). During H2 or when slices were kept in 4 mmol/L glucose, AV vanished even more quickly, but the times to block did not differ significantly between slices from controls and mutants. After reoxygenation, AV fully recovered in most slices. Rates of blockade of fEPSPs were comparable in all slices, and most fEPSPs recovered fully after HI. But even in the presence of 10 mmol/L glucose, the second hypoxia suppressed fEPSPs irreversibly in some slices: 2 of 10 from control, 3 of 7 from mdx, and 1 of 6 from mdx3cv mice. Most slices in 4 mmol/L glucose showed no recovery at all: six of seven from control, three of five from mdx, and four of five from mdx3cv mice. Thus, slices from mdx mice were more susceptible than other slices to irreversible hypoxic failure when slices were kept in 10 mmol/L glucose, but they were less susceptible than other slices when kept in 4 mmol/L glucose. In conclusion, the lack of full-length dystrophin (427 kDa) predisposes to quicker loss of nerve conduction in slices from mdx and mdx3cv mutants and improved posthypoxic recovery of fEPSPs in 4 mmol/L glucose in slices from mdx but not mdx3cv mutants, perhaps because the 70-kDa and other C-terminal isoforms are still present in mdx mice.

2-Deoxyglucose-induced long-term potentiation in CA1 is not prevented by intraneuronal chelator.

In hippocampal slices, temporary (10-20 min) replacement of glucose with 10 mM 2-deoxyglucose is followed by marked and very sustained potentiation of EPSPs (2-DG LTP). To investigate its mechanism, we examined 2-DG's effect in CA1 neurons recorded with sharp 3 M KCl electrodes containing a strong chelator, 50 or 100 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). In most cases, field EPSPs were simultaneously recorded and conventional LTP was also elicited in some cells by tetanic stimulation of stratum radiatum. 2-DG potentiated intracellular EPSP slopes by 48 +/- 5.1% (SE) in nine cells recorded with plain KCl electrodes and by 52 +/- 6.2% in seven cells recorded with EGTA-containing electrodes. In four of the latter cells, tetanic stimulation (twice 100 Hz for 1 s) failed to evoke LTP (2 +/- 1.1%), although field EPSPs were clearly potentiated (by 28 +/- 6.9%). Thus unlike tetanic LTP, 2-DG LTP is not readily prevented by postsynaptic intraneuronal injection of EGTA. These findings agree with other evidence that the rise in postsynaptic (somatic) [Ca(2+)](i) caused by 2-DG is not the principal trigger for the subsequent 2-DG LTP and that it may be a purely presynaptic phenomenon.

Early effects of hypoxia on brain cell function.

This article reviews the changes in neuronal function produced by oxygen lack, especially as observed in hippocampal slices in vitro. An early cessation of electrical activity ("firing"), caused by a K+ conductance-mediated neuronal hyperpolarization and disappearance of excitatory synaptic potentials (EPSPs), can be seen as a protective mechanism that prevents the cellular damage resulting from severe mismatch between energy needs and supplies. These changes are triggered by such hypoxia-induced signals as a rise in cytoplasmic free calcium, fall in adenosine triphosphate (ATP), and extracellular accumulation of adenosine (produced by ATP breakdown). Upon reoxygenation, the suppression of neuronal/synaptic activity is quite reversible, as long as hypoxic nerve cells have an adequate supply of glucose. But if sufficient ATP cannot be obtained by anerobic glycolysis to maintain essential Na-K pump activity and protein synthesis, long-term cell functi on and survival are compromised. Thus, when both oxygen and glucose are deficient, as in strokes, the cellular protective mechanisms cannot prevent the lethal effects of excessive Ca2+ influx.

Diabetes mellitus preserves synaptic plasticity in hippocampal slices from middle-aged rats.

Synaptic plasticity is generally believed to provide a cellular mechanism for learning and memory. One manifestation of synaptic plasticity, long-term potentiation in the CA1 region, was compared in hippocampal slices from young and older rats, both control animals and streptozotocin-treated diabetics with moderate hyperglycemia ( approximately 15 mM). Long-term potentiation of excitatory synaptic potentials, elicited by tetanic stimulation or by 2-deoxy-D-glucose application, was readily obtained in slices from young (four-month-old) control and diabetic rats, but not in slices from middle-aged (12-month-old) control rats. Both forms of potentiation, however, could be elicited in slices from 12-month-old diabetics. The unexpected finding that long-term potentiation is restored in moderately hyperglycemic older rats suggests that the blood glucose level of older animals may be important for synaptic plasticity and perhaps for the ability to learn.

Persistent block of CA1 synaptic function by prolonged hypoxia.

The effects of prolonged hypoxia were studied by field and intracellular recordings from hippocampal slices of the rat, kept submerged at 34 degrees C. When artificial cerebrospinal fluid contained 10 mM glucose, even very long exposures to hypoxia or 300 microM cyanide (21-25 min) did not block field excitatory postsynaptic potentials and population spikes irreversibly. By contrast, in the presence of 4 mM glucose, hypoxia lasting only 9-13 min-ending 2-3 min after the characteristic transient recovery ("hypoxic injury potential")-resulted in irreversible block of synaptic responses. Voltage-dependent sodium channels and N-methyl-D-aspartate receptors are involved, because irreversible block was prevented by tetrodotoxin (0.5 microM), kynurenate (2 mM) or DL-aminophosphonovalerate (50 microM), whereas 6,7-dinitroquinoxaline-2,3-dione (25 microM) suppressed only the transient recovery. The hypoxic suppression of afferent volleys in slices kept in 4 mM glucose was also prevented by kynurenate or aminophosphonovalerate. Intracellular recordings revealed opposite effects of hypoxia according to glucose concentration: in 10 mM glucose, mainly hyperpolarization; in 4 mM glucose, after a brief hyperpolarization, a major and usually irreversible depolarization. In the presence of kynurenate or tetrodotoxin, major depolarizations also occurred, but they were reversible. Thus, large depolarizations of hippocampal neurons do not necessarily lead to irreversible block of synaptic transmission: there is lasting damage only when hypoxia is combined with low glucose, presumably because a reduced supply of glycolytically generated ATP limits the Na+/K+ pump's ability to maintain or restore membrane potentials and thus prevent excessive activation of N-methyl-D-aspartate receptors.

Cocaine and lidocaine have additive inhibitory effects on the GABAA current of acutely dissociated hippocampal pyramidal neurons.

Inhibition mediated by gamma-aminobutyric acid (GABA) is a major target for the central actions of cocaine and lidocaine, which can result in seizures, especially when these drugs are abused in combination. In the present study, we investigated how cocaine and lidocaine interact to depress GABA current (IGABA), recorded by the whole-cell technique in freshly isolated rat hippocampal neurons. Cocaine depressed IGABA in a concentration dependent manner, such that cocaine was more potent against lower than higher GABA concentrations: the cocaine IC50 was 0.13, 0.62 and 1.2 mM for GABA at 2, 10 and 100 microM, respectively. Cocaine depressed IGABA to the same extent in the absence and presence of 1 microM tetrodotoxin, indicating that cocaine inhibition of IGABA is distinct from its Na+ channel blocking action. Lidocaine reversibly depressed IGABA evoked by 10 microM GABA, with an IC50 of 9.8 mM. In the presence of 3 mM lidocaine, 0.3 mM cocaine depressed IGABA (10 microM GABA) to 30+/-7%. The significantly greater depression by the combined agents (p<0.05) indicates additive effects on the GABA receptor/channel complex, which are likely to contribute to the additive convulsant effects noted when these drugs are abused in combination.

Cocaine decreases the glycine-induced Cl- current of acutely dissociated rat hippocampal neurons.

The effects of cocaine on glycine-induced Cl- current (I(GLY)) of single neurons, freshly isolated from the rat hippocampal CA1 area, were studied with conventional whole-cell recording under voltage-clamp conditions. Cocaine depressed I(GLY) in a concentration-dependent manner, with an IC50 of 0.78 mM. Preincubation with 1 mM cocaine alone had no effect on I(GLY), suggesting that resting glycine channels are insensitive to cocaine. The depression of I(GLY) by cocaine was independent of membrane voltage. Internal cell dialysis with 1 mM cocaine failed to modify I(GLY). Because the depression of I(GLY) was noncompetitive, cocaine may act on the glycine receptor-chloride ionophore complex at a site distinct from that to which glycine binds. The cocaine suppression of I(GLY) was unaffected by 1 microM tetrodotoxin and 1 microM strychnine. Blockers of protein kinase C (Chelerythrine), kinase A (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide HCl, (H-89)) and Ca-calmodulin-dependent kinase (1-[N,O-bis(5-isoquinoline-sulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperaz ine (KN-62)) were also ineffective, which suggests that these phosphorylating mechanisms do not modulate cocaine-induced suppressant action on I(GLY). This extracellular, strychnine-independent depression of I(GLY) may contribute to cocaine-induced seizures.

Intraneuronal [Ca2+] changes induced by 2-deoxy-D-glucose in rat hippocampal slices.

Temporary replacement of glucose by 2-deoxyglucose (2-DG; but not sucrose) is followed by long-term potentiation of CA1 synaptic transmission (2-DG LTP), which is Ca2+-dependent and is prevented by dantrolene or N-methyl--aspartate (NMDA) antagonists. To clarify the mechanism of action of 2-DG, we monitored [Ca2+]i while replacing glucose with 2-DG or sucrose. In slices (from Wistar rats) kept submerged at 30 degreesC, pyramidal neurons were loaded with [Ca2+]-sensitive fluo-3 or Fura Red. The fluorescence was measured with a confocal microscope. Bath applications of 10 mM 2-DG (replacing glucose for 15 +/- 0.38 min, means +/- SE) led to a rapid but reversible rise in fluo-3 fluorescence (or drop of Fura Red fluorescence); the peak increase of fluo-3 fluorescence (DeltaF/F0), measured near the end of 2-DG applications, was by 245 +/- 50% (n = 32). Isosmolar sucrose (for 15-40 min) had a smaller but significant effect (DeltaF/F0 = 94 +/- 14%, n = 10). The 2-DG-induced DeltaF/F0 was greatly reduced (to 35 +/- 15%, n = 16) by,-aminophosphono-valerate (50-100 microM) and abolished by 10 microM dantrolene (-4.0 +/- 2.9%, n = 11). A substantial, although smaller effect, of 2-DG persisted in Ca2+-free 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA) medium. Two adenosine antagonists, which do not prevent 2-DG LTP, were also tested; 2-DG-induced DeltaF/F0 (fluo-3) was not affected by the A1 antagonist 8-cyclopentyl-3, 7-dihydro-1,3-dipropyl-1H-purine-2,6-dione (DPCPX 50 nM; 287 +/- 38%; n = 20), but it was abolished by the A1/A2 antagonist 8-SPT; 25 +/- 29%, n = 19). These observations suggest that 2-DG releases glutamate and adenosine and that the rise in [Ca2+] may be triggered by a synergistic action of glutamate (acting via NMDA receptors) and adenosine (acting via A2b receptors) resulting in Ca2+ release from a dantrolene-sensitive store. The discrepant effects of sucrose and 8-SPT on DeltaF/F0, on the one hand, and 2-DG LTP, on the other, support other evidence that increases in postsynaptic [Ca2+]i are not essential for 2-DG LTP.

Higher sensitivity of CA1 synapses to aglycemia in streptozotocin-diabetic rats is age-dependent.

We studied conduction velocity in peripheral nerves and the block of synaptic transmission produced by lack of glucose in hippocampal slices from 4- and 12-month-old streptozotocin-induced diabetic rats and their age-matched controls. In sural nerves of young and old diabetic rats, the conduction velocity was reduced by 30-35%. In slices from young diabetics, CA1 synaptic transmission was more sensitive to aglycemia than in control slices. However, all slices from older rats showed comparable increases in CA1 synaptic sensitivity to aglycemia. We conclude that the cerebral adaptation to diabetic hyperglycemia apparent in the hippocampus of young rats is masked in older rats by an age-dependent increase in sensitivity to lack of glucose.

Potassium conductance causing hyperpolarization of CA1 hippocampal neurons during hypoxia.

In experiments on slices (from 100- to 150-g Sprague-Dawley rats) kept at 33 degreesC, we studied the effects of brief hypoxia (2-3 min) on CA1 neurons. In whole cell recordings from submerged slices, with electrodes containing only KMeSO4 and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and in the presence of kynurenate and bicuculline (to minimize transmitter actions), hypoxia produced the following changes: under current clamp, 36 cells were hyperpolarized by 2.7 +/- 0.5 (SE) mV and their input resistance (Rin) fell by 23 +/- 2.7%; in 30 cells under voltage clamp, membrane current increased by 114 +/- 22.3 pA and input conductance (Gin) by 4.9 +/- 0.9 nS. These effects are much greater than those seen previously with K gluconate whole cell electrodes, but only half those seen with "sharp" electrodes. The hypoxic hyperpolarizations (or outward currents) were not reduced by intracellular ATP (1-5 mM) or bath-applied glyburide (10 microM): therefore they are unlikely to be mediated by conventional ATP-sensitive K channels. On the other hand, their depression by internally applied ethylene glycol-bis-(beta-aminoethyl ether)-N,N, N',N'-tetraacetic acid (1.1 and 11 mM) and especially 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (11-33 mM) indicated a significant involvement of Ca-dependent K (KCa) channels. The beta-adrenergic agonist isoprenaline (10 microM) reduced hypoxic hyperpolarizations and decreases in Rin (n = 4) (and in another 11 cells corresponding changes in Gin); and comparable but more variable effects were produced by internally applied 3':5'-adenosine cyclic monophosphate (cAMP, 1 mM, n = 6) and bath-applied 8-bromo-cAMP (n = 8). Thus afterhyperpolarization-type KCa channels probably take part in the hypoxic response. A major involvement of G proteins is indicated by the near total suppression of the hypoxic response by guanosine 5'-O-(3-thiotriphosphate) (0. 1-0.3 mM, n = 23) and especially guanosine 5'-O-(2-thiodiphosphate) (0.3 mM, n = 26), both applied internally. The adenosine antagonist 8-(p-sulfophenyl)theophylline (10-50 microM) significantly reduced hypoxic hyperpolarizations and outward currents in whole cell recordings (with KMeSO4 electrodes) from submerged slices but not in intracellular recordings (with KCl electrodes) from slices kept at gas/saline interface. In further intracellular recordings, antagonists of gamma-aminobutyric acid-B or serotonin receptors also had no clear effect. In conclusion, these G-protein-dependent hyperpolarizing changes produced in CA1 neurons by hypoxia are probably initiated by Ca2+ release from internal stores stimulated by enhanced glycolysis and a variable synergistic action of adenosine.

In rat hippocampal slices, NMDA receptor-mediated EPSPs are more sensitive to hypoxia than AMPA receptor-mediated EPSPs.

In slices kept at 33 degrees C, N-methyl-D-aspartate (NMDA) receptor- and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated field excitatory post-synaptic potentials (EPSPs) were pharmacologically isolated in CA1. Both types of EPSPs were reversibly blocked by 3 min of hypoxia (95% N2/5% CO2); but NMDA receptor-mediated EPSPs were consistently blocked earlier and recovered later than AMPA receptor-mediated EPSPs, recorded in the same slice. This difference may be due to inactivation of NMDA receptors by hypoxia-induced acidity and/or rise in internal [Ca2+].

2-Deoxy-D-glucose-induced changes in membrane potential, input resistance, and excitatory postsynaptic potentials of CA1 hippocampal neurons.

Temporary block of glycolysis by 2-deoxy-D-glucose (2-DG) reversibly suppresses synaptic transmission in the CA1 region of hippocampal slices. Recovery of responses is followed by a sustained potentiation of field excitatory postsynaptic potentials (EPSPs) (2-DG-LTP). To investigate the mechanisms involved in this type of LTP, we studied the effects of 2-DG on membrane properties of CA1 neurons (in slices from Sprague-Dawley rats), recorded with sharp intracellular electrodes containing 3 M KCl, as well as patch electrodes, filled mainly with 150 mM KMeSO4 and Hepes. The predominant change produced by 15- to 20-min applications of 2-DG (10 mM, replacing glucose) was hyperpolarization (-5.6 +/- 1.1 mV for 18 intracellular recordings and -7.2 +/- 0.80 mV for 17 whole-cell recordings) accompanied by a fall in resistance (-33 +/- 2.5% for 14 intracellular recordings and -11.6 +/- 7.1% for 15 whole-cell recordings). Virtually identical hyperpolarizations were recorded in the presence of 20 microM glyburide (-5.5 +/- 1.5 mV, n = 6), but they were abolished by adenosine antagonists 8-(p-sulfophenyl)theophylline (8-SPT) and 8-cyclopentyl-3,7-dihydro-1,3-dipropyl-1H-purine-2,6-dione (DPCPX) (2.8 +/- 1.6 and 4.0 +/- 1.7 mV, respectively; n = 5 for both). It was concluded that the hyperpolarization is most likely caused by an increase in K+ conductance, activated by a 2-DG-induced rise in adenosine release. After such applications of 2-DG, a sustained potentiation of EPSPs (similar to the 2-DG-LTP of field EPSPs) was evident in five neurons recorded with intracellular electrodes but not in any of nine whole-cell recordings, where it was replaced by sustained, LTD-like depression. We conclude that a factor essential for 2-DG-LTP induction is lost during whole-cell recording.